Zeitschrift für Nephrologie und Therapeutik

Production of Thrombin Complexes with DNA Aptamers Containing G-Quadruplex and Different Duplexes

Abstract

Vera A. Spiridonova *,Glinkina KA ,Gainutdinov AA ,Arutyunyan AM

Despite major advances in the understanding of the molecular mechanisms of thrombosis and the development of effective thrombolytic agents, arterial thrombosis remains a formidable clinical problem. A new class of direct thrombin inhibitors (aptamers), has been described recently. Aptamers, single-stranded oligonucleotides with a length of 30-60 nucleotides, exhibit high affinity and specificity towards any defined recognition target (protein). Aptamers are antibody analogs in terms of both specificity and affinity, with an apparent advantage of the former to being reproduced by anautomated chemical synthesis. Aptamers are routinely selected by SELEX technology (Systematic Evolution of Ligands by Exponential enrichment), which is thoroughly discussed elsewhere. The DNA aptamers to thrombin have a very highly ordered tertiary structure (G-quadruplex). A great number of the DNA aptamers to thrombin with sophisticated structure have been designed and widely investigated as potential thrombin inhibitors using traditionalmedicine tests (TT, PT, APPT). In this manuscript we suggest the different structures of aptamers, based on RE31 structure, so-called G-quadruplex, which are bound to short and long duplex. The RE31 aptamer had being previously shown to have an order of magnitude prolonged thrombintime in comparison to 15TBA aptamer. The purpose of the present study was to investigate the trunked aptamers to thrombin structures by CD spectroscopy and estimate the stability of the thrombin complexes with aptamers using electrophoresis in polyacrilamide gels. Our findings clearly show that both G-quadruplex and duplex domains of RE31 as well as trunked aptamers are strong effectors of aptamer complex stability. Using a set of oligonucleotide models derived from RE31 sequence, we have shown that the attached duplex domain of trunked aptamers retains the antiparallel unimolecular G-quadruplex topology seen for 15TBA.